Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA.

Integrated retroviral DNA is flanked by short direct repeats of the target DNA. The length of these repeats is specific for the provirus that is integrated (H.E. Varmus, in J.A. Shapiro, ed., Mobile Genetic Elements, 1983). For the human immunodeficiency virus type I (HIV-1), the length of the direct repeats in the target DNA was shown to be 5 bp in one case (Muesing et al., Nature [London] 313:450-458, 1985) and 7 bp in another (Starcich et al., Science 227:538-540, 1985). One possible explanation for this discrepancy is that the direct repeats flanking HIV-1 proviruses are variable. To investigate this, we analyzed the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. In each clone the provirus was flanked by a 5-bp direct repeat of human DNA. Analysis of the proviral clone previously described as being flanked by a 7-bp direct repeat of target DNA (Starcich et al., op. cit.) revealed that this clone was flanked by a 5-bp repeat instead. Therefore, we conclude that HIV-1 proviruses are flanked by 5-bp direct repeats of human DNA. The sequences of the 5-bp duplications from the different proviral clones do not have any apparent similarity to each other or to HIV-1 DNA.     

Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes.

All presently available replication-competent proviral clones of human immunodeficiency virus type 1 (HIV-1) are derived from cell culture-amplified virus. Since tissue culture is highly selective for viral strains with an in vitro growth advantage, such clones may not be representative of the biologically relevant virus present in vivo. In this study, we report the molecular cloning and genotypic characterization of 10 HIV-1 genomes directly from uncultured brain tissue of a patient with AIDS dementia complex. Targeting unintegrated circular HIV-1 molecules for recombinant lambda phage cloning, we obtained four full-length genomes with one or two long terminal repeats (LTRs), three defective genomes with internal deletions, two rearranged genomes with inverted LTR sequences, and one integrated proviral half with flanking cellular sequences. Nucleotide sequence analysis of these clones demonstrated chromosomal integration, circle formation, genomic inversion, and LTR-mediated autointegration of HIV-1 genomes in vivo. Comparison of a 510-bp hypervariable envelope region among 8 lambda phage-derived and 12 polymerase chain reaction-derived clones from the same brain specimen identified a predominant viral form as well as genetically divergent variants. Variability among 19 of 20 clones ranged between 0.2 and 1.2%. One clone exhibited 8.2% nucleotide sequence differences consisting almost exclusively of G-to-A changes. Transfection of the four full-length HIV-1 genomes identified one clone (YU-2) as replication competent and exhibiting growth characteristics similar to those of tissue culture-derived macrophage tropic strains of HIV-1. These results demonstrate, for the first time, that replication-competent HIV-1 genomes, complex mixtures of defective viral forms, and chromosomally integrated provirus persist in vivo. In addition, the brain-derived viral clones are expected to prove valuable for future studies of macrophage and neurotropism as well as for the analysis of other viral properties that are subject to in vitro selection pressures.     

Repeat arrays in cellular DNA related to the Epstein-Barr virus IR3 repeat.

We isolated clones and determined the sequence of portions of mouse and human cellular DNA which cross-hybridize strongly with the IR3 repetitive region of Epstein-Barr virus. The sequences were found to be tandem arrays of a simple sequence based on the triplet GGA, very similar to the IR3 repeat. The cellular repeats have distinct differences from the viral repeat region, however, and their sequences do not appear capable of being translated into a purely glycine-plus-alanine protein domain like the portion of the Epstein-Barr nuclear antigen coded by IR3. Although the relationship between IR3 and the cellular repeats is left unclear, the cellular repeats have many interesting features. The tandem arrays are about 1 to several kilobases long, much shorter than satellite tandem repeats and larger than other interspersed, tandem repeats. Each of the repeats is a distinct variation, perhaps diverged from a common sequence, (GGA)n. This family is present in the genomes of all species tested and appears to be a ubiquitous feature of all higher eucaryotic genomes.     

JC virus regulatory region tandem repeats in plasma and central nervous system isolates correlate with poor clinical outcome in patients with progressive multifocal leukoencephalopathy

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has a hypervariable regulatory region (JCV RR). A conserved archetype form is found in the urines of healthy and immunocompromised individuals, whereas forms with tandem repeats and deletions are found in the brains of PML patients. Type I JCV RR, seen in MAD-1, the first sequenced strain of JCV, contains two 98-bp tandem repeats each containing a TATA box. Type II JCV RR has additional 23-bp and 66-bp inserts or fragments thereof and only one TATA box. We cloned and sequenced JCV RR from different anatomic compartments of PML patients and controls and correlated our findings with the patients' clinical outcome. Twenty-three different sequences were defined in 198 clones obtained from 16 patients. All 104 clones with tandem repeats were type II JCV RR. Patients with poor clinical outcome had high proportions of JCV RR clones with both tandem repeats in plasma (54%) and brain or cerebrospinal fluid (85%). In those who became survivors of PML, archetype sequences predominated in these anatomic compartments (75 and 100%, respectively). In patients with advanced human immunodeficiency virus infection without PML, only 8% of JCV RR clones obtained in the plasma contained tandem repeats. These data suggest that the presence of tandem repeats in plasma and CNS JCV RR clones is associated with poor clinical outcome in patients with PML.     

Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.     

Molecular characterization of an attenuated human immunodeficiency virus type 2 isolate.

Naturally occurring strains of human immunodeficiency virus (HIV) can vary considerably in their in vitro biological properties, and such differences may also be reflected in their in vivo pathogenesis. In an attempt to define genetic determinants of viral pathogenicity, we have molecularly cloned, sequenced, and characterized an attenuated isolate of HIV type 2 (HIV-2/ST) that differs from prototype HIV-2 strains in its inability to fuse with and kill susceptible CD4-bearing target cells. A proviral clone, termed JSP4-27, was identified to be transfection competent and to fully exhibit the noncytopathic and nonfusogenic properties of its parental isolate. Nucleotide sequence analysis of this clone revealed a genomic organization very similar to that of cytopathic HIV-2 strains and an overall nucleotide sequence homology of 88 to 90%. Amino acid sequence comparison confirmed the integrity of all major viral gene products in JSP4-27 but identified two amino acid sequence substitutions in its envelope fusion region. To investigate whether these mutations were responsible for the nonfusogenic phenotype of JSP4-27, we amplified, cloned, and sequenced the envelope fusion regions of four additional HIV-2/ST strains, two of which represented in vitro-generated, fusogenic and cytopathic variants of HIV-2/ST. The analysis showed that all HIV-2/ST strains examined, including the fusogenic variants, contained the same amino acid sequence changes. On the basis of these findings, we conclude that the attenuated phenotype of JSP4-27, and that of its parental virus, is not due to a direct alteration of the envelope fusion domain. Our results also show, for the first time, that individual replication-competent proviral clones can be representative of attenuated strains of HIV.     

A novel minisatellite at a cloned hamster telomere

The ends of eukaryotic chromosomes have special properties and roles in chromosome behavior. Selection for telomere function in yeast, using a Chinese hamster hybrid cell line as the source DNA, generated a stable yeast artificial chromosome clone containing 23 kb of DNA adjacent to (TTAGGG)n, the vertebrate telomeric repeat. The common repetitive element d(GT)n appeared to be responsible for most of the other stable clones. Circular derivatives of the TTAGGG-positive clone that could be propagated in E. coli were constructed. These derivatives identify a single pair of hamster telomeres by fluorescence in situ hybridization. The telomeric repeat tract consists of (TTAGGG)n repeats with minor variations, some of which can be cleaved with the restriction enzyme MnlI. Blot hybridization with genomic hamster DNA under stringent conditions confirms that the TTAGGG tracts are cleaved into small fragments due to the presence of this restriction enzyme site, in contrast to mouse telomeres. Additional blocks of (TTAGGG)n repeats are found 4–5 kb internally on the clone. The terminal region of the clone is dominated by a novel A-T rich 78 bp tandemly repeating sequence; the repeat monomer can be subdivided into halves distinguished by more or less adherence to the consensus sequence. The sequence in genomic DNA has the same tandem organization in probably a single primary locus of >20–30 kb and is thus termed a minisatellite.     

Molecular cloning of MuLV proviruses integrated into the genome of mouse erythroleukemia cells. I. Characteristics of endogenous proviruses

The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60-70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.     

Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit

The human polymorphic epithelial mucin (PEM) is expressed apically by glandular epithelium and by the carcinomas that develop from these tissues. Previously isolated cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 60 bp tandem repeats (TR), making it an expressed minisatellite. We now report the full genomic sequence of the PEM gene, including 803 bp of 5' flanking sequence. The gene is composed of 7 exons and varies in size from approximately 4 to approximately 7 kb, depending on the number of tandem repeats in exon 2. Expression of PEM was obtained from a genomic clone in an Epstein-Barr virus based vector, after transfection into a human epithelial cell line, indicating the presence of effective regulatory sequences in this clone.     

Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Delta132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Delta132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Delta132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.     

分子克隆化禽网状内皮组织增生症病毒传染性及其前病毒全基因组序列研究

利用脂质体转染技术,将含有SNV株禽网状内皮组织增生症病毒 （REV）前病毒全基因组cDNA克隆质粒转染鸡胚成纤维细胞（CEF）.用对REV的单克隆抗体和抗REV env-gp90的鼠血清作间接免疫荧光反应,在原始的转染细胞及随后传代的细胞中均显示病毒特异性抗原.而且,在连续传代细胞中的阳性率明显升高.用REV特异性引物对进一步传代后的细胞基因组作PCR,也检测出REV基因组.这些结果均表明所得到的分子克隆化病毒具有传染性,因而也进一步证明所用的质粒克隆包含有具感染性的全病毒基因组.对该全基因组cDNA克隆进行酶切所获得的数个亚克隆进行测序,并将序列进行拼接,完成了REV全基因组序列.REV的这个传染性克隆将有助于进一步研究REV的分子生物学特性.     

Comparison of the complete DNA sequences of the Oka varicella vaccine and its parental virus

The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.     

DNA of noninfectious and infectious integrated spleen necrosis virus (SNV) is colinear with unintegrated SNV DNA and not grossly abnormal.

The cleavage sites of eight restriction endonucleases in linear spleen necrosis virus (SNV) DNA were mapped, and the map was oriented with respect to viral RNA. With the aid of this map, several structural features of the viral DNA were elucidated: unintegrated linear SNV DNA is terminally redundant; the majority of SNV DNA molecules integrated in chicken DNA, which were previously shown to be present in many sites in cellular DNA, are colinear with unintegrated viral DNA; no tandem integration of proviral molecules is detectable; and the majority of integrated SNV DNA molecules, including integrated SNV DNA molecules previously shown to be noninfectious, do not have an altered restriction enzyme digestion pattern.     

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.